The problem of progression of renal fibrosis leading to endstage renal failure remains unresolved. The progressive tubulointerstitial lesions are characterized by tubuloepithelial damage and atrophy, peritubular capillary rarefication and interstitial fibrosis with leukocyte infiltrates. Work from the PI's has provided evidence that the leukocyte infiltrate is chemokine dependent and contributes to the progressive fibrosis. The present proposal will focus on factors contributing to the generation of chemokines and cytokines at the site of injury and to the resulting leukocyte infiltration and fibrosis. We will examine the hypothesis: Hypoxia and the generation of Damage Associated Molecular Pattern (DAMP) molecules occur at sites of chronic tubulointerstitial damage. Hypoxia will activate HIF transscription factors and DAMP molecules will activate Toll-like receptors(TLR) both on epithelial cells and on residentmonocyte/macrophages(M/M). HIF and TLR activation will generate cytokines and chemokines which will in turn determine the specific type of infiltrating bone marrow (b.m.) derived cells. Together with direct local activation of M/M by HIF and TLRs, the infilux of b.m. derived cells will determine the further outcome of the disease process. The specific aims are to identify the contribution of epithelial cells and of b.m. derived cells activated by hypoxia via HIF and by DAMP molecules via TLRs to the outcome of tubulointerstitial fibrosis secondary to glomerular disease. This will be achieved by a combination of in vitro and in vivo as well as ex vivo-in vivo experiments using conditional knockouts for two HIF isoforms, HIF-11 and 21, as well as knockouts for TLR2 and 4. The in vivo models to be used are adriamycin nephrosis and collagen 4A3 deficient -Alport mice. Both models develop secondary interstitial disease with leukocyte infiltration and fibrosis. We will also attempt to identify the specific chemokine receptors involved in the recruitment of specific subsets of myeloid cells to the tubulointerstitial lesions developing in these models by using chemokine deficient myeloid cells. The combination of these experiments should provide us with important and novel information on the role of HIF and TLRs as sensors for chronic renal injury and that of chemokine chemokine receptors as effectors for the infiltration of specific populations of b.m.derived cells in the process of progressive interstitial fibrosis. The concept of hypoxia generating DAMP molecules, which inturn act as intrinsic TLR activators may open up a totally new perspective on mechanisms of progression of renal diseases. Obviously this information is prerequsite for designing future strategies to interfere with progressive renal fibrosis. PUBLIC HEALTH RELEVANCE End stage renal disease is a major public health problem with insufficient therapies at present. We plan to identify new disease pathways that will allow the design of novel therapies.